Matthew A. Roberts

UV laser machined polymer substrates for the development of microdiagnostic systems

This report describes a UV laser photoablation method for the production of miniaturized liquid-handling systems on polymer substrate chips. The fabrication of fluid channel and reservoir networks is accomplished by firing 200 mJ pulses from an UV excimer laser at substrates moving in predefined computer-controlled patterns. This method was used for producing channels in polystyrene, polycarbonate, cellulose acetate, and poly(ethylene terephthalate). Efficient sealing of the resulting photoablated polymer channels was accomplished using a low-cost film lamination technique. After fabrication, the ablated structures were observed to be well defined, i.e., possessing high aspect ratios, as seen by light, scanning electron, and atomic force microscopy. Relative to the original polymer samples, photoablated surfaces showed an increase in their hydrophilicity and rugosity as a group, yet differences were noted between the polymers studied. These surface characteristics demonstrate the capability of generating electroosmotic flow in the cathodic direction, which is characterized here as a function of applied electric field, pH, and ionic strength of common electrophoretic buffer systems. These results show a correlation between the ablative changes in surface conditions and the resulting electroosmotic flow. The effect of protein coatings on ablated surfaces is also demonstrated to significantly dampen the electroosmotic flow for all polymers. All of these results are discussed in terms of developing liquid-handling capability, which is an essential part of many μ-TAS and chemical diagnostic systems.

Electrochemical Detection in Polymer Microchannels

A method, using UV laser photoablation, is presented for the fabrication and the integration of an electrochemical detector in a microchannel device, where carbon microband electrodes are placed either in the bottom or in the side walls of the rectangular microchannel. The different electrochemical cell geometries are tested with a model compound (ferrocenecarboxylic acid) in 40- and 100-μm-wide capillaries fabricated in planar polymer substrates. The experimental results are compared to numerical simulations for stagnant stream conditions. Depending on the scan rate and on the microchannel depth, the system behaves as a microband electrode until a linear diffusion field develops within the channel. The limit of detection for a one electron redox species within the 120-pL detection volume is ∼1 fmol with both cyclic voltammetry and chronoamperometric detection.

Liposome Behavior in Capillary Electrophoresis

The behavior of liposomes in capillary electrophoresis is studied for the purpose of developing a potential method for characterizing liposomes prepared for use in industrial and analytical applications. This study characterizes the electrophoretic behavior of liposomes under various conditions to provide information about electrophoretic mobility and liposome-capillary surface interactions. The results of this method are compared with the results obtained using traditional laser light-scattering methods to obtain size information about liposome preparations. Additionally, reactions of liposomes and the surfactant n-octyl-β-d-glucopyranoside are performed off-line in bulk solution experiments and on-line in the capillary. Automated delivery of lysis agents by multiple electrokinetic injections is demonstrated as a general method for inducing on-capillary reactions between liposomes and other reagents. Furthermore, some preliminary evidence on the use of liposomes as a hydrophobic partitioning medium for analytical separations is presented.

Improved liposome immunomigration strip assay for alachlor determination

The feasibility of a simple, single-use immunomigration strip assay for alachlor was previously demonstrated. In the device, capillary action caused alachlor and alachlor-tagged, dye-containing liposomes to migrate through an anti-alachlor antibody zone, on a plastic-backed nitrocellulose strip, where competitive binding occurred. Unbound liposomes continued migration to a liposome capture zone, where they were quantified by densitometry. The amount of liposome-entrapped dye measured in this zone was directly proportional to the alachlor concentration in the sample. This report describes modifications to various components of the system, leading to improvement in the sensitivity of the assay to the point where the maximum contaminant level of alachlor, 2 ppb, can be easily detected. Measurements of liposome size and antibody cross-reactivity are also presented. The new methodology involves acid treatment of the antibody and the use of preincubation of the analyte-tagged liposomes, free analyte and anti-alachlor before initiating migration. This results in strong interactions between the anti-alachlor, liposome and strip such that liposomes that have bound to antibody do not migrate. This inhibition of migration is reversed by free analyte, which competes with the liposome for the available antibody binding sites. As in the previous assay, unbound liposomes migrate to a capture zone where they can be quantified, and the color intensity of this zone is directly proportional to the amount of analyte present. This technique produces an assay capable of detecting alachlor at levels down to 1 ppb.

Flow-injection liposome immunoanalysis (FILIA) for alachlor.

An automated Flow-Injection Liposome ImmunoAnalysis (FILIA) system has been modified from a previous design, using a specific environmental contaminant, the herbicide alachlor, as a model analyte. Signal amplification by means of fluorescent marker-loaded, analyte-tagged liposomes provides high sensitivity. The computer controlled system is composed of commercially available components, with the exception of the column packing material, which has to be prepared for each specific analyte to be determined. The use of such components means that the system is easily modified. The relationships between antibody concentration and assay speed and sensitivity are explored, and the possibilities of using the system for determination of multiple analytes is discussed.

Liposome immunosensing devices for environmental contaminant screening

Abstract Single-use strip assays using immunomigration and immunoabsorption formats have been developed for the extra-laboratory determination of environmental contaminants. Dye-loaded liposomes are used as the amplification strategy in these immunoassays. Details of the assay performance and liposome characteristics are given.

Investigation of a Novel Microtiter Plate Support Material and Scanner Quantitation of Immunoassays, Proteins and Phospholipids

Abstract A recently developed shallow-well microtiter plate, made from a specially formulated polymer that binds proteins, peptides and nucleic acids rapidly and efficiently, has been investigated for use in solid-phase immunoassays as well as for protein and phospholipid assays. The assay uses quantitation of the color intensity of a dye on the solid phase by means of a simple desk-top scanner coupled to a computer which allows the gray-scale density of the color to be easily and accurately measured. Consequently, this approach is independent of the absorption spectrum of the dye used. These studies demonstrate that the novel technology incorporated into these unique shallow-well microtiter plates has potential in both protein and immunoassays. In the latter case, there are considerable savings in both time and material costs over conventional ELISA methods. The scanning software provided for the solid-phase immunoassay is user configurable for other densitometry assays on different solid matrices and ca...

Immunoassay Devices for Extra-Laboratory Measurements of Toxic Chemicals Based on Capillary Migration and Liposome Amplification

Simple, single-use biosensing devices are being developed based on the immunologic recognition of specific environmental and food commodity contaminants in combination with signal amplification strategies using marker-loaded liposomes to provide high sensitivity. Solution flow in the devices is controlled by capillary action and detection can be based on marker color or electroactivity. Several areas of investigation are being addressed: 1) immobilization of antibodies onto the solid phase of the capillary bed; 2) liposome surface density of analyte molecules conjugated to phospholipids; 3) capillarity-driven timing sequence for optimum flow and interaction of components; 4) concentration/measurement zone based on an avidin-capture mechanism; and 5) evaluation of several modes of detection/quantitation. Preliminary laboratory testing of several prototypes of this device based on optical detection has demonstrated the feasibility of this approach.